Methods for purifying camptothecin compounds

ABSTRACT

Methods for purifying camptothecin compounds are described. The camptothecin compounds in purified form are useful in the treatment of cancer.

Field of the Invention

The present invention relates to methods for purifying camptothecin(CPT) compounds.

20(S)-Camptothecin is a natural alkaloid product which is present as acomponent in a crude plant material isolated from the Chinese treeCamptotheca acuminata (Nyssaceae) (Wall, M.; Wani, M. C.; Cooke, C. E.;Palmer, K. H.; McPhail, A. T.; and Slim, G. A., "Plant antitumor agents.I. The isolation and structure of camptothecin, a novel alkaloidalleukemia and tumor inhibitor from Camptotheca acuminata", J. Am. Chem.Soc. 88:3888-3890, 1966). CPT can also be isolated from the Indian treeMappie foetida Miers (Govindachari, T. R. and Viswanathan, N.,"Alkaloids of Mappia Foetida", Phytochemistry 11:3529-3531 (1972)). CPThas been determined to possess considerable antitumor activity (Wani, M.C. and Wall, M. E. "Plant Antitumor Agents. II. The Structure of Two NewAlkaloids from Camptotheca acuminata", J. Org. Chem. 34(5):1364-1367(1968) and has since been the focus of much scientific endeavor (Seereviews: 1) Shamma, M.; St. Georgiev, V.; Journal of PharmaceuticalSciences 63(2):163-183 (1974); 2) D'Arpa, P. and Liu, L. F."Topoisomerases-targeting antitumor drugs" Biochimica et BiophysicaActa. 989:163177 (1989); 3) Corcoran, J. W. and Hahn, F. E. "Mechanismof Action of Antimicrobial and Antitumor Agents" Antibiotics, Vol. III,Springer-Verlag, New York, Heidelberg, Berlin, pp. 48-57 (1975); 4)Hutchinson, C. R. "Camptothecin: Chemistry, Biogenesis and MedicinalChemistry", Tetrahedron 37:1047-1065 (1981); 5) Cai, J. C. andHutchinson, C. R., "Camptothecin", The Alkaloids, Vol. XXV, pp. 101-137(1983)). Recently, it was shown that CPT has high activity against humancancer xenografts in vivo (Giovanella, B. C.; Hinz, H. R.; Kozielski, A.J., Stehlin, Jr., J. S.; Silber, R. "Complete Growth Inhibition of HumanCancer Xenografts in Nude Mice by Treatment with 20(S)-Camptothecin",Cancer Research, 51:3052-3055 (1991)), as well as in vitro (Pantazis,P.; Hinz, H. R.; Mendoza, J. T.; Kozielski, A. J.; Williams, Jr., L. J.;Stehlin, Jr., J. S.; Giovanella, B. C. "Complete Inhibition of GrowthFollowed by Death of Human Malignant Melanoma Cells In Vitro andRegression of Human Melanoma Xenografts in Immunodeficient Mice byCamptothecins," Cancer Research, 52:3980-3987 (1992)). It is of interestthat a highly efficient method be developed for the purification of CPTsince CPT is generally commercially available only as a plant extract.

The traditional methods of purifying CPT involve the use of conventionalchromatography using a mixture of solvents, usually a nonpolar solventin conjunction with a polar solvent to affect the separation ofindividual molecules from each other with silica gel, followed bycrystallization from methanol-acetonitrile. (Wall, M. E., FourthInternational Symposium on the Biochemistry and Physiology of Alkaloids,Halle, DDR, Jun. 25-28, 1969, pp. 77-87; Wall et al. 1966, supra.)

SUMMARY OF THE INVENTION

An object of the present invention is to provide novel methods forpurifying camptothecin compounds, preferably 20(S)-Camptothecin.

Additional objects and advantages of the present invention will be setforth in part in the description which follows, and in part will beapparent from the description, or may be learned by practice of thepresent invention. The objects and advantages of the present inventionwill be realized and attained by means of the elements and combinationsparticularly pointed out in the appended claims.

To achieve the objects and in accordance with the purpose of the presentinvention, as embodied and broadly described herein, the presentinvention relates to a method of purifying camptothecin, preferably20(S)-Camptothecin, comprising the steps of (1) charging a column withan adsorbent compound and wetting the compound with a non-polar solvent;(2) passing a non-polar solvent containing a starting materialcontaining CPT through the column; (3) eluting the column with anon-polar solvent, whereby components of the starting materialcontaining CPT both more polar and less polar than the CPT are elutedand wherein the non-polar solvent in steps (1), (2) and (3) can be thesame or different; (4) eluting the column with a solvent more polar thanthe non-polar solvent(s); and (5) separating the CPT from the eluentresulting from the elution in step (4) to obtain the purified CPT.

Further, the present invention relates to a method of purifying astarting material containing CPT comprising the steps of: (1) heatingunder reflux a mixture of the starting material containing CPT and anexcess amount of acetic acid under conditions whereby a mixture free ofoxygen is formed; (2) adding HBr, HCl or a mixture thereof to themixture in an effective amount and continuing said heating under refluxuntil the starting material containing CPT is dissolved; (3) monitoringthe heating under reflux by means of thin layer chromatography andcontinuing the heating until the material immediately following CPT on athin layer chromatography plate has disappeared; (4) removing plant anddecomposition products from the solution; and (5) separating CPT fromthe solution by crystallization to obtain the purified CPT.

Also, the present invention relates to a method of purifying a startingmaterial containing CPT comprising the steps of: (1) converting saidstarting material containing CPT to a derivative thereof containing asubstituent on the C20-hydroxyl group thereof; (2) purifying thederivative as set forth above; and (3) converting the derivative back toCPT to obtain the purified CPT.

In addition, the present invention relates to a method of purifying astarting material containing CPT comprising the steps of: (1) combiningan aqueous base, organic solvent, and the starting material containingCPT to form an aqueous phase and an organic phase; (2) separating theorganic phase from the aqueous phase; (3) acidifying the aqueous phase;and (4) separating the crystals obtained in step (3) to obtain thepurified CPT.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the present invention, as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1a and 1b show the effectiveness of CPT purified in accordancewith the present invention when administered intramuscularly againsthuman breast cancer.

FIGS. 2a and 2b show the results obtained by treating a human bladdercancer xenograft by intramuscular administration of CPT purified inaccordance with the present invention.

FIGS. 3a and 3b show the same results for human prostate cancer.

FIGS. 4a and 4b show the same results for human leukemia.

FIGS. 5a and 5b show the same results for human colon cancer.

FIGS. 6a and 6b show the same results for human osteosarcoma.

FIGS. 7a and 7b show the same results for human pancreatic cancer.

FIGS. 8a and 8b show the same results for human melanoma cancer.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT(S)

CPT is an alkaloid component of a crude plant material isolated fromCamptothera acuminata. The crude plant material can be obtained, forinstance, from the Institute of Materia Medica, Academia Sinica,Shanghai, China, or Sigma Biochemicals, or Good Land Enterprises,Vancouver, Canada.

The natural plant material--regardless of its source--does not containonly CPT, but also other natural products, e.g. derivatives of CPT,which have to be separated from CPT. For instance, the plant materialhas also the presence of 10-hydroxy-20(S)-CPT, 10-methoxy-20(S)-CPT,11-hydroxy-20(S)-CPT and 11-methoxy-20(S)-CPT (Wani, M. C. and Wall, M.E., 1968) and also 20-deoxycamptothecin ((a) Hsu, J.-S.; Chao, T.-Y.;Lin, L.-T.; Hsu, C.-F., Hua Hsueh Hsueh Pao 1977, 35:193; Chem. Abstr.1979, 90:28930; (b) Adamovics, J. A.; Cina, J. A.; Hutchinson, C. R.Phytochemistry, 18:1085 (1979); (c) Lin, L.-T.; Sung, C.-C.; Hsu, J.-S.,K'o Hsueh Tung Pao, 24(10:478-479 (1979); Chem. Abstr. 1979, 91:193482u)in the starting material containing CPT from camptotheca acuminata. Thestarting material containing CPT may also contain 9-methoxy-20(S)-CPTand mappicine, if the source of the material is Mappia Foetida Miers(Olacaceae) (Govindachari, T. R. and Viswanathan, N., "9-Methoxycamptothecin. A New Alkaloid from Mappia foedia Miers", Indian Journalof Chemistry, 10(1):453-454 (1972); and Govindacharie, T. R.;Ravindranath, K. R.; Viswanathan, J. "Mappicine, a Minor Alkaloid fromMappia foetida Miers"), 10-methoxy-20(S)-CPT, if the source isOphiorrhiza mungos (Rubiaceae) (Tafur, S.; Nelson, J. D.; DeLong, D. C.;Svoboda, G. H. "Antiviral Components of Ophiorrhiza mungos- Isolation ofCamptothecin and 10-Methoxycamptothecin" Lloydia 39:261-262 (1976)), or9-methoxy-20(S)-CPT if the source is Ervatania heyneana (Gunasekera, S.P.; Badawi, M. B.; Cordell, G. A.; Farnsworth, N.R.; Chitis, M. "PlantAnticancer Agents X. Isolation of Camptothecin and 9-Methoxycamptothecinfrom Ervatamia Heyneana", Journal of Natural Products, 42(5):475-477(1979)).

It is to be understood that starting material containing CPT as usedherein refers to crude CPT, CPT plant extract, any plant materialcontaining CPT, synthetic CPT, reaction products containing CPT, and anyCPT-containing material which can be purified by any one of the methodsof the present invention. It is also further understood that purifiedCPT as used herein means CPT which has a purity greater than the purityof the starting material containing CPT.

Thus, CPT, in order to be used in anticancer activity should beextensively purified prior to administering because: (1) the naturalproduct contains several other components which contribute to a largedegree of toxicity, for example in mice, and (2) FDA regulations requiresuch purifying for any drug or compound.

Method I: Purification of CPT by Means of Column Chromatography

In this method, a column, such as any commercially availablechromatography column, for example, columns made by Ace Glass or FisherScientific, having for example the dimensions 5×90 cm, but may besmaller or larger, depending on the amount of material to bechromatographed, is charged with a non-polar solvent along with anadsorbent compound, e.g., a silica gel, alumina, magnesium silicate,magnesia, carbon, diatomaceous earth, porous beads, and specialadsorbents or a combination thereof obtained for example from Aldrich orSigma. Any column may be used which allows the adsorbent to be packedinside the column (composed of plastics, Pyrex® or other forms or glassor glass-like materials, pure metals or suitable alloys of metals oralloys of metals with nonmetals, steel or stainless steel or a suitablecombination thereof known and understood by those skilled in the art) ina fashion such that the sample to be chromatographed may be introducedat one end of the adsorbent and then separated into one or moreindividual components and eluting through the other end of the column byflowing solvent through the packed adsorbent. This process may becarried out at low pressures (less than one atmosphere) up to very highpressures of several thousand atmospheres of pressure known to thoseskilled in the art, including, but not limited to, HPLC. The separationmay also be carried out at normal room temperature or higher forexample, 100° C., or lower for example, 0° C., temperatures. A preferrednon-polar solvent is CH₂ Cl₂. Other non-polar solvents can also be used,such as ethers, hexanes, pentanes, CHCl₃ and so forth.

The adsorbent, e.g. silica or alumina, which is used generally has asize of 70-270 mesh. Other grades may also be used. The amount ofnon-polar solvent and the adsorbent, is directly related to the size ofthe column which is used for the purification. For example, in a columnhaving the dimensions 5×90 cm, approximately 200 ml of adsorbent isused.

Starting material containing CPT is stirred into a non-polar solventwhich contains an adsorbent. Optionally, a little sand may be added tothe top of the adsorbent, such as silica gel, after the adsorbentsettles. After the adsorbent settles, substantially all of the non-polarsolvent is decanted and more non-polar solvent is added almost to thetop of the adsorbent bed and sand line, if sand is used. The adsorbentcontaining most of the CPT is then added to the top of the column,followed by the supernatant liquid containing the rest of the startingmaterial containing CPT.

Generally, for every 1000 ml of non-polar solvent, for example CH₂ Cl₂,used, approximately 150 ml of absorbent are used.

Upon adding the starting material containing CPT in the non-polarsolvent, the column is then developed or eluted with fresh non-polarsolvent which can be the same or different from the non-polar solventsdiscussed above, for example, CH₂ Cl₂.

The progress of the column development can be monitored by suchtechniques as thin layer chromatography (TLC), which uses a two to threepercent concentration of CH₃ OH in CHCl₃.

In this method, it is most remarkable to note that not only the lesspolar material clears the column before the more polar CPT, but alsothat other components, which are more polar than CPT, actually surpassCPT and clear the column before CPT does. After the clearance of theseless polar and more polar components, CPT may then be retrieved by theaddition and elution with a solvent or mixture of solvents more polarthan the non-polar solvent(s) used. Examples of polar solvents includemethanol, ethanol, propanol, isopropanol and so forth. Thus, thepurified CPT is retrieved by eluting the column with the said polarsolvent. The purified CPT in the solution may then subsequently berecrystallized by various known methods to those skilled in the art suchas slow evaporation of the solvent which contains the purified CPT.Another means of recrystallization which is part of the presentinvention is the recrystallizing from N,N-dimethylacetamide (DMA), orany other suitable medium that has the ability to bind acidic hydroxylhydrogens. Further, an additional recrystallization step can be usedwherein the recrystallization is from acetic acid/ethanol by dissolvingthe crystals in hot acetic acid and then adding ethanol as described inmethod II below, which at this point removes further impurities from thecrystals.

The manner in which CPT behaves with regard to movement on silica in anon-polar medium contributes to the ease of purification of not onlyCPT, but also the efficient isolation of the various other componentspresent in the plant material.

Method II: Isolation of CPT Chemical Treatment and Recrystallization forLarge Quantity Purification of CPT

In this method, acetic acid and starting material containing CPT arecharged into a container, such as a round bottom flask. Generally, forevery 100 grams of starting material containing CPT, 2500 ml of aceticacid are used. Upon charging the acetic acid and starting materialcontaining CPT into the container, oxygen is removed therefrom, such asby heating the mixture to about 100° C. for approximately one hour undera continuous stream of nitrogen or any inert gas. After this point, HBr,HCl or a mixture thereof is added to the container. For example, 100 mlof 48% aqueous HBr and 200 ml concentrated HCl can be used.

The mixture is heated, generally for about six hours. Though notnecessary, it is preferred that when the temperature reachesapproximately 100° C. during heating, another aliquot of HBr, HCl or amixture thereof, e.g. 50 ml HBr, is added to the solution and theheating is continued until all of the starting material containing CPTis dissolved. At this point, the temperature of the solution may beapproximately 106° C. during this continuing refluxing. The heating iscontinued until the material immediately following the CPT (backspot) ona TLC plate (using a mixture of 3% CH₃ OH:CHCl₃ as the eluant) hasdisappeared. Typically, in a 3% CHOH:CHCl₃ system, the frontspot has anR_(f) value of 0.7. The "backspot" has an R_(f) value of 0.6 and thephenolic 10-hydroxy CPT, 9-hydroxy CPT or 11-hydroxy CPT has an R_(f)value of 0.1. Afterwards, purified CPT is separated from the solution.In one preferred embodiment, water is added. Generally, approximately350 ml of water per 2500 ml of acetic acid are added to the solution.Upon adding water, though not necessary, it is preferred that thesolution be placed in a lower temperature atmosphere such as arefrigerator for about 12 hours. Afterwards, the solution is filtered.Such filtering can be accomplished by a buchner funnel with a smallfritt pore to remove plant and decomposition products from the solution,or by using a filter aid such as Celite®. To the resulting supernatant,ice-water is added which results in the crystallization of purified CPTwhich is collected on the filtering device, such as a buchner funnel.The crystals should then be washed, for instance, with water, until thewash is no longer acidic to pH paper. The crystals are then broken upand dried overnight in a vacuum by, for example, applying a vacuum ofapproximately 0.1 atmosphere and heating the crystals to about 50° C.Alternatively, the crystals may be allowed to air dry in a hood.

At this point, as judged by TLC, the crystals may still contain some ofthe more polar phenolic compounds and other less polar compounds, asvisualized by TLC. The phenolic compounds may be removed byrecrystallization from N,N-dimethylacedemine (DMA) (or any othersuitable medium that has the ability to remove the acidic hydroxylhydrogen-containing compounds like 9-, 10- or 11-hydroxy CPT). A furtherstep in the purification may be another recrystallization from aceticacid/ethanol (the composition of this mixture is described in Example 2infra.), which at this point, removes the less polar materials from thecrystals. Trituration from absolute ethanol yields slightly yellow CPT,which is analytically pure on TLC and has a minimum purity of 98% asestablished by HPLC and ¹ H-NMR. The supernatant may be concentrated invacuo and the resulting crystals may be re-recrystallized to obtain afurther crop of CPT. The quantities purified by this method may bescaled up indefinitely and seem to be only limited by the size of thereaction vessels.

Method III: Purification of CPT by Chemical Derivatization Followed byIsolation of the Derivative and the Subsequent Reconversion to CPT

Another route of chemical modification/isolation of CPT is to firstconvert CPT to a more soluble and better behaved molecule. This iseasily achieved by transforming CPT into a derivative, which has a muchbetter solubility than CPT, and therefore makes it much easier toisolate by standard purification procedures. Such a modification of CPTmay be carried out at the C₂₀ -hydroxyl group, by converting CPT into aderivative for instance, an ester or ether. One such derivative may befor example an acetate like the one described by Wall et al. (1966)supra., or any other, as long as the goals are accomplished, forinstance, greater solubility in organic solvents combined with arelatively easy reconversion of the derivative back to CPT. An esterwith these properties is easily obtainable and purifiable by any of theusual chromatographic procedures known to those skilled in the art, suchas the procedure used in Example 3, infra. After the ester is purifiedby removal of most of the impurities, the ester may be hydrolyzed by anynumber of hydrolysis methods known to those skilled in the art to giveback CPT in a pure form. This may be accomplished, for example, byheating the respective ester in aqueous sulfuric acid or acetic acid inthe presence of HCl and/or HBr as set forth in Example 3, infra. Afterthe reconversion is complete, the CPT is recrystallized by the methodusing acetic acid/ethanol as described earlier in method II.

It should be pointed out that many different derivatives may besynthesized by modification of the C₂₀ hydroxyl group. This derivativeis not at all limited to esters, although esters are preferred.

The same holds true for the hydrolysis of the derivative to yield backCPT in a purified form. Many different media, including organic andinorganic acids and bases can be used in this hydrolysis step.

Method IV: Purification of CPT by Converting the Lactone Ring to theCarboxylate Salt, Followed by Removal of By-Products and Reconversion toNatural CPT

Another method which works quite effectively involves the chemicalmodification of the lactone ring system in CPT. The lactone ring islabile to basic conditions, allowing it to be readily converted to itscorresponding carboxylate salt by treatment with a base, e.g. onecontaining an hydroxide ion, which allows the effective conversion andextraction of CPT into the aqueous phase. By conducting this chemicalderivatization, most of the other natural products and plant productsare removed by being retained in the organic phase. Reconversion of thesalt form to the lactone ring containing CPT-compounds is accomplishedby reacidification of the aqueous layer, for example, by HCl and/or HBror other suitable acids and subsequent isolation of the original, nativeform of CPT. Successive recrystallizations of the CPT material asdiscussed above yields the same pure material as stated earlier.

The present invention will be further clarified by the followingExamples, which are intended to be purely exemplary of the presentinvention.

EXAMPLE 1

A column obtained from Ace Glass (5×90 cm) was charged with a nonpolarsolvent, CH₂ Cl₂ and 200 ml of silica of 70-270 mesh. Starting materialcontaining CPT (10 gm) recorded as 93% CPT pure was stirred into asolution containing 1000 ml CH₂ Cl₂ and 150 ml of silica gel. Aftersettling, substantially all the solvent was decanted. The silicacontaining most of the CPT was added to the top of the column, followedby the supernatant solution containing the rest of the CPT. The columnwas then developed with pure CH₂ Cl₂. The progress of the development ofthe column was monitored by TLC, which used a two to three percentconcentration of CH₃ OH in CHCl₃. Not only the less polar materialscleared the column before CPT, but also the other components, which weremore polar than CPT on silica, actually surpassed the CPT and clearedthe column before the CPT. After the clearance of these less polar andmore polar components, CPT was retrieved when a polar solvent, methanol,was added to the CH₂ Cl₂.

The yield as a result of this purification method was 4 gm. The puritywas 99% by TLC.

EXAMPLE 2

In a well ventilated fume hood, a 5000 ml round bottom flask wasequipped with a reflux condenser, nitrogen inlet and thermometer andcharged with 2500 ml of acetic acid and 100 gm of starting materialcontaining CPT (recorded as 93.7% CPT) obtained from Good LandEnterprise, Vancouver, Canada, and heated for one hour under acontinuous stream of nitrogen in order to flush all the oxygen out ofthe solution. After this point, 100 ml of 48% aq. HBr and 200 ml conc.HCl were added and the reaction mixture was heated for 6 hours, afterwhich another aliquot of 50 ml HBr was added to the refluxing mixture.The temperature was monitored and when it reached approximately 100° C.,another aliquot of 50 ml HBr was added to the solution and heating wascontinued until all the CPT dissolved. At this point the temperature ofthe solution was approximately 106° C. The heating was continued untilthe material immediately following CPT on a TLC plate (using a mixtureof 3 % CH₃ OH : CHCl₃ as the eluant) disappeared. At this point 350 mlof H₂ O was added and the solution was put into a refrigerator for about12 hours. The solution was then filtered through a large buchner funnelwith a small fritt pore in order to remove plant and decompositionproducts from the solution. 2000 ml of ice-water were then added to thesupernatant and after refrigerating the mixture overnight, the resultingcrystals were collected on a buchner funnel and washed with a largeamount of water until the wash was no longer acidic.

The resulting CPT product was then recrystallized as follows.

To one gram of this CPT-product mixture 15 ml of DMA were added to abeaker wrapped with thermoinsulating material. This mixture wasmagnetically stirred and heated to 140°-145° C. during which the CPTmaterial went into the solution. The solution was filtered hot through abuchner funnel and allowed to cool slowly to room temperature and thenput into the refrigerator. The crystals formed in this manner werecollected on a buchner funnel and the material was re-recrystallizedusing 13 ml of DMA until the orange color phenolic material disappearedon TLC when checked with a 3% CH₃ OH:CHCl₃ solvent system. The yield was65% CPT.

The crystalline material obtained in this fashion was re-recrystallizedfrom acetic acid:ethanol to remove the less polar material in thefollowing manner: One gram of this CPT was put into a flask and 28 ml ofacetic acid was added and the mixture was heated until all the CPT wasdissolved. At this point, 13 ml of ethanol were slowly added and thesolution was then allowed to cool to room temperature very slowly toenhance the crystal purity. This step is repeated if the TLC system (3%CH₃ OH:CHCl₃) still showed the presence of this less polar front spot.The yield of CPT was 0.46 gm and the purity was 99%.

EXAMPLE 3

20 gm of starting material containing CPT (recorded as 93% CPT pure)were added under an inert atmosphere to 400 ml of pyridine catalyst at0° C. and 23 ml of acetic anhydride. The solution was stirred for anhour at this temperature and then allowed to reach room temperature. Thereaction was monitored by thin layer chromatography (TLC) using 3%methanol:chloroform as the eluant. After completion of the reaction, thepyridine was removed in vacuo and 200 ml of methylene chloride wasadded. The organic phase of the solution was then washed with 1N HCl,followed by water and dried with anh. Na₂ SO₄ sulfate. The organic phaseof the solution was then adsorbed onto a column with silica and anelution was carried out with CH₂ Cl₂. All of the less polar fractionsand the one backspot component immediately trailing CPT were removed asdescribed in method I. After removal of these compounds the polarity ofthe solvent was changed to 3% MeOH:CH₂ Cl₂. Approximately 17 gm ofacetate were obtained by this method contaminated with a little CPT.Hydrolysis was carried out by heating, under nitrogen atmosphere, onegram of the acetate with 10 ml 40% HBr and 5 ml H₂ O. The reactionprogress was monitored by TLC and after two hours, the reaction wascomplete. The mixture was cooled and 80 ml water was added toprecipitate all the CPT out of the solution. The crystals were washedwith water to remove all the hydrobromic acid followed byrecrystallization from acetic acid:ethanol as described in method II.The yield of CPT was 0.9 gm and the purity was 99%.

EXAMPLE 4

1 gm of crude plant extract obtained from Good Land Enterprises(Vancouver, Canada) (recorded as 93% CPT) was added to 150 ml of CHCl₃and 50 ml of 1% NaOH at low temperature (-5° C. to 0° C.). The mixturewas stirred for 60 to 120 minutes at this temperature. The solution wasfiltered through a buchner funnel containing a bed of Celite® andtransferred to a separatory funnel. The aqueous phase was separated andthe organic phase was shaken once with 10 ml of ice cold 1% NaOH. Theaqueous phases were then combined and washed once with 10 ml CHCl₃ andacidified slowly with ice cold 6M HCl and allowed to stand in therefrigerator. The resulting precipitate was filtered off. The yellowmaterial was free of the backspot. The yield was 0.91 grams and thepurity was less than 99%. As outlined in method II, this material wasrecrystallized from 20 ml DMA followed by re-recrystallization fromacetic acid:ethanol. The yield was 0.44 grams and purity was 99%.

In order to show how the purified CPT of the present invention can beused, for example, in anti-cancer activity, the following example wasconducted.

EXAMPLE 5

Swiss nude mice of the NIH high fertility strain were bred andmaintained pathogen-free in the laboratory of the Stehlin Foundation.(Giovanella, B. C. and Stehlin, J. S. "Heterotransplantation of humanmalignant tumors in `nude` thymusless mice. I. Breeding and maintenanceof `nude` mice." J. Natl. Cancer Inst. 51: 615-619; 1973.)

Human malignant carcinomas of the colon, breast, lung, ovary, stomach,and malignant melanomas were heterotransplanted directly from a patientinto the nude mice and passaged serially. In particular, the tumors CLO,MUR, CAS, SW 48, SQU and BRO as described in Giovanella, B. C., Stehlin,J. S., Shepard, R. C. and Williams, L. J. "Correlation between responseto chemotherapy of human tumors in patients and in nude mice." Cancer52: 1146-1152; 1983), and tumor SCH as described in Heim, S., Mandahl,N., Arheden, K., Giovanella, B. C., Yim, S. O., Stehlin, J. S., Jr. andMitelman, F. "Multiple karyotypic abnormalities including structuralrearrangements of 11P in cell lines from malignant melanomas." CancerGenet. Cytogent. 35: 5-20; 1988, and Verschraegen, C., Giovanella, B.C., Mendoza, J. T., Kozielski, A. J. and Stehlin, J. S., Jr. "Specificorgan metastases of human melanoma cells injected into the arterialcirculation of nude mice." Anticancer Res., In Press were used. TumorsCLO (Breast Carcinoma), BOL (Bladder Carcinoma), PC-3 (ProstateCarcinoma), BM #3 (Leukemia), WIL (Colon Carcinoma), TEP (Osteosarcoma),LOV (Pancreatic Carcinoma), BRO (Melanoma) were heterotransplanted. Forthe experiments, the tumor tissue was finely minced in complete MEMmedium and 0.5 ml of a 10% v/v suspension was inoculated subcutaneouslyon the upper back of groups of 10-30 mice. When the tumors becamepalpable and measurable in all the animals, they were divided intogroups of 4-8 and treated with the desired dose of the drug inexperiment or with the vehicle only for the controls.

CPT purified by method II was prepared for IM-injection or oraladministration using cottonseed oil for example which included thedispersion of the test compound by sonication at 60° C. for 1 hour atthe standard concentration of 0.25 to 1 mg/ml using an UltrasonicProcessor, Model 2200 by Branson Ultrasonics Corp., Danbury Conn.06810-1961. Intramuscular injections were performed through a 27-gaugeneedle into the deep muscles of the posterior legs of the mice twice aweek. The animals received up to 70 such injections consecutivelywithout suffering ill effects except for some local fibrosis.

Oral administration was achieved by injecting the required amounts ofdrugs suspended in cottonseed oil directly into the stomach. FIGS. 1aand 1b show the effectiveness of CPT purified by one method of thepresent invention against the human breast cancer when administeredintramuscularly, showing that even prolonged intramuscularadministration of the compound of the present application do not haveany effect on the body weight of the mice at the dosage of 2.5 mg/kgusing cottonseed oil as the carrier. FIGS. 2a and 2b show the resultsobtained by treating a human bladder cancer xenograft by intramuscularadministration of CPT purified by one method of the present invention.

FIGS. 3a and 3b show the same results for human prostate cancer.

FIGS. 4a and 4b show the same results for human leukemia.

FIGS. 5a and 5b show the same results for human colon cancer.

FIGS. 6a and 6b show the same results for human osteosarcoma.

FIGS. 7a and 7b show the same results for human pancreatic cancer.

FIGS. 8a and 8b show the same results for human melanoma cancer.

It is clear from these studies, that purified CPT with the closedlactone ring have been demonstrated to possess an astonishing level ofanticancer activity. This applies both to the spectrum of tumors coveredand to the quality of the responses. The CPT purified by the methods ofthe present invention has been able to block growth completely and tototally regress human xenografts of carcinomas (lungs, breast, colon,prostate, leukemia, osteosarcoma, pancreatic, bladder) and malignantmelanomas. This has been accomplished without any observable toxicity.

Other embodiments of the present invention will be apparent to thoseskilled in the art from consideration of the specification and practiceof the present invention disclosed herein. It is intended that thespecification and examples be considered as exemplary only, with a truescope and spirit of the invention being indicated by the followingclaims.

What is claimed is:
 1. A method of purifying 20(S)-camptothecincomprising the steps:(1) combining an aqueous base containing anhydroxide ion, a water immiscible organic solvent, and a startingnatural plant material containing 20(S)-camptothecin to form a mixturehaving an aqueous phase containing 20(S)-camptothecin and an organicphase, wherein the lactone ring in said 20(S)-camptothecin is convertedto a carboxylate salt in the aqueous phase; (2) separating said organicphase from said aqueous phase containing 20(S)-camptothecin; (3)acidifying said aqueous phase containing 20(S)-camptothecin to form20(S)-camptothecin crystals by the addition of an acid whereby thecarboxylate salt is reconverted to said lactone ring; and (4) separating20(S)-camptothecin crystals obtained in step (3).
 2. The method of claim1, further comprising the step of adding an aqueous base to the organicphase separated in step (2) to form an organic phase and an aqueousphase therein; then separating said aqueous phase from said organicphase and combining it with the aqueous phase separated in step (2). 3.The method of claim 1, wherein said starting material containing20(S)-camptothecin is plant material selected from the group consistingof Camptotheca acuminata, Mappie foetida Miers, Ophiorrhiza mungos, andErratama heyneana.
 4. The method of claim 1, wherein said aqueous baseis sodium hydroxide.
 5. The method of claim 1, wherein saidwater-immiscible organic solvent is chloroform.
 6. The method of claim1, wherein said acid is HCl, HBr, acetic acid, H₂ SO₄, HI, or mixturesthereof.
 7. The method of claim 6, wherein said acid is HCl and/or HBr.8. The method of claim 1, wherein step (4) is accomplished by filteringthe resulting crystals from step (3).